Chapter 3 immobilized enzymes

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<ul><li><p>ImmobilizationImmobilized biocatalysts can be made from killed cells, cell fragments, purified enzymes, and respiring cellsDefinition: Enzymes or cells which are physically confined to a defined region in space while retaining their catalytic activity and have the ability to be repeatedly and continuously used</p></li><li><p>Immobilized Enzyme SystemsEnzyme Immobilization:</p><p>- Easy separation from reaction mixture, providing the ability to control reaction times and minimize the enzymes lost in the product. </p><p>- Re-use of enzymes for many reaction cycles, lowering the total production cost of enzyme mediated reactions.</p><p>- Ability of enzymes to provide pure products. </p><p>- Possible provision of a better environment for enzyme activity</p><p>- Diffusional limitation </p></li><li><p>Higher dilution rates can be used in a CSTR. Easier product recovery. Lowered viscosity.Easier enzyme recovery.</p><p>Why.Advantages of Immobilization</p></li><li><p>Mass transfer is inhibitedReduced activity of enzymesBursting of immobilization medium due to cell growthCells/enzymes can leak out of immobilization medium Disadvantages of Immobilization</p></li><li><p>Immobilization MethodsPhysicalChemical</p></li><li><p>Membranes</p></li><li><p>Membrane MicroencapsulationMembrane polymerized around aqueous enzyme solution in colloidal suspension (particle sizes on the order of 100-10 m)</p><p>Organic solventAqueous enzyme solutionmixingAdd polymer</p></li><li><p>MicroencapsulationAdvantagesHigh surface to volume ratioThin membraneRelatively benign attachment methodProblemsMore easily damaged than gel beads</p></li><li><p>Other Membrane Immobilization Methods- HFMHollow fiber reactors use micro- or ultra-filtration membranes to retain high MW enzymes, but pass low MW compoundsEnzymes or cellsSubstrate and products outside the membraneSubstrate diffuses through membraneProduct diffuses out of the membrane</p></li><li><p>Close-up view of fiber</p></li><li><p>Hollow Fiber ReactorEnzymes or cells inSubstrate inProduct out</p></li><li><p>Two Types of Cartridges</p></li><li><p>Entrapment</p></li><li><p>Entrapment Immobilization is based on the localization of an enzyme within the lattice of a polymer matrix or membrane. - retain enzyme - allow the penetration of substrate. </p><p>It can be classified into matrix and micro capsule types. Immobilized Enzyme Systems</p></li><li><p>Immobilized Enzyme SystemsEntrapment - Matrix Entrapment- Membrane Entrapment(microencapsulation)</p></li><li><p>EntrapmentAdvantagesRelatively benign attachment methodEasy to performProblemsMass transfer limitationsCell/enzyme leakageSome cell/enzyme deactivationAlginate (polymer) common entrapment matrix</p></li><li><p>Immobilized Enzyme SystemsMatrix Materials: Organics: polysaccharides, proteins, carbon, vinyl and allyl polymers, and polyamides. e.g. Ca-alginate, agar, K-carrageenin, collagenImmobilization procedures:Enzyme + polymer solution polymerization extrusion/shape the particles</p><p>Inorganics: activated carbon, porous ceramic.</p><p>Shapes: particle, membrane, fiber</p></li><li><p>Immobilized Enzyme SystemsEntrapmentchallenges:- enzyme leakage into solution</p><p>- diffusional limitation</p><p>- reduced enzyme activity and stability</p><p>- lack of control micro-environmental conditions.</p><p>It could be improved by modifying matrix or membrane.</p></li><li><p>Covalent Bonding</p></li><li><p>Immobilized Enzyme SystemsSurface immobilizationAccording to the binding mode of the enzyme, this method can be further sub-classified into: - Physical Adsorption: Van der WaalsCarriers: silica, carbon nanotube, cellulose, etc. Easily desorbed, simple and cheap, enzyme activity unaffected.</p><p>- Ionic Binding: ionic bonds Similar to physical adsorption.Carriers: polysaccharides and synthetic polymers having ion-exchange centers.</p></li><li><p>Immobilized Enzyme SystemsSurface immobilizationCovalent Binding: covalent bonds Carriers: polymers contain amino, carboxyl, sulfhydryl, hydroxyl, or phenolic groups.</p><p> - Loss of enzyme activity - Strong binding of enzymes </p></li><li><p>Covalent BondingMost extensively used method of immobilization due to high bond strengthThree elements- structural polymer, enzyme molecule and bridge moleculeActivity of an immobilized enzyme is a strong function of the hydrophilicity of the structural polymer</p></li><li><p>Covalent Bonding To SupportBridging molecule needs to be small, have two reactive groups, and not bond at the active site</p></li><li><p>Immobilized Enzyme SystemsCross-linking is to cross link enzyme molecules with each other using agents such as glutaraldehyde.</p><p>Features: similar to covalent binding.</p><p>Several methods are combined.</p></li><li><p>Copolymerization of EnzymesCopolymerization is performed with multifunctional bridge molecules to yield and insoluble productUsually an inert protein is also included</p></li><li><p>Chemistry of Covalent Immobilization</p></li><li><p>Adsorption</p></li><li><p>Immobilization by AdsorptionBinding forces are ionic, hydrophobic, hydrogen bonds, or Van der Waals interactionsBinding is simple (stir together in a beaker) but is reversible. Substrate addition can cause desorption.</p></li><li><p>Typical AdsorbentsCellulosePolystyrene resinsKaoliniteGlassAluminaSilica gel</p></li></ul>